K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANYRat, Mouse and Human
GLP-1 EIA
For the quantitative determination of GLP-1
in rat, mouse and human plasma.

Cat. No. KT-384

For Research Use Only. Not for Use in Diagnostic Procedures.

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K-ASSAY 

KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATION

Rat, Mouse and Human GLP-1 EIA
Cat. No. KT-384
INTENDED USE
The Rat, Mouse and Human GLP-1 EIA is for the quantitative determination of GLP-1 in rat, mouse and human plasma.
For research use only, not for use in diagnostic procedures.

INTRODUCTION
GLP-1 is a peptide hormone from the intestinal mucosa, which is produced from its precursor, proglucagon, by posttranslational processing. The mammalian proglucagon is synthesized in the neuroendocrine L-cell of the intestine and thealpha-cells of the pancreas. It contains within its structure the sequences of glucagon and two glucagon-like peptides(GLP-1 and GLP-2) in tandem flanked at their amino and carboxyl termini by dibasic residues. GLP-1 is a 37 amino acidpeptide and is produced in the human small intestine and pancreas, in either C-terminal-amidated or glycine-extendedform.
GLP-1 (7-36) amide and its receptor are present in several brain regions and may play a role in the physiological controlof feeding. Several reports have been presented as follows as to the biological activities of GLP-1. GLP-1 (7-37) and (736) amide is known as one of the most potent insulin secretagogues.
GLP-1 (7-36) amide was supposed to improve glycemic control in patients with type 2 diabetes by increasing insulin
secretion, by inhibiting glucagon secretion and by delaying gastric draining rather than by altering extrapancreatic glucose
metabolism. Intravenous GLP-1 (7-37) and (7-36) amide could normalize fasting hyperglycemia in type 2 diabetic patients.
Hyperglycemia during parenteral nutrition could be controlled by exogenous GLP-1, whereas the chronic therapy of type 2
diabetes required GLP-1 derivatives with longer duration of action. Recombinant GLP-1 (7-36) amide was recently shownto cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion,
5-day treatment of hereby obese human subjects with GLP-1 at high doses by prandial subcutaneous infusion promptly
slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and reduced foodintake with an ensuing weight loss.
A G-protein-coupled receptor, GPR120, which is abundantly expressed in the intestine, functions as a receptor forunsaturated long-chain FFAs (free fatty acids). The stimulation of GPR120 by FFAs promotes the secretion of GLP-1 invitro (measured by KT-384) and in vivo, and increases circulation insulin, indicate that GPR120-mediated GLP-1 secretioninduced by dietary FFAs is important in the treatment of diabetes.
All these approaches have shown remarkable efficacy in both experimental and clinical studies. The GLP-1-based therapyof type 2 diabetes, therefore, represents a new and attractive alternative. Advantages of this assay include highsensitivity, high specificity and no interference from other components in plasma samples. The GLP-1 calibrator of this kit
is a highly purified synthetic product.

PRINCIPLE
This EIA kit is based on a competitive enzyme immunoassay using a highly specific antibody combined with a biotinavidin affinity system. The 96 well plate is coated with goat anti-rabbit IgG and GLP-1 Calibrators or samples, biotinylated
human GLP-1 and GLP-1 antibody are added to the wells for a competitive immuno-reaction. After incubation and rinsing
excess GLP-1, HRP-labeled streptoavidins (SA-HRP) are added to bind to the antigen-antibody complex so that HRPlabeled streptoavidin-biotinylated GLP-1-antibody complexes are formed on the surface of the wells. Finally, HRP enzyme
activity is determined by o-Phenylenediamine dihydrochloride (OPD) and the concentration of GLP-1 is calculated.

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KAMIYA BIOMEDICAL COMPANYCOMPONENTS
Component

Form

1. Antibody-Coated Plate
2. GLP-1 Calibrator
3. Labeled Antigen
4. GLP-1 Antibody
5. SA-HRP
6. SA-HRP Diluent
7. Substrate Buffer
8. OPD Tablet
9. Stop Solution
10. Buffer Solution
11. Wash Solution
Concentrate
12. Plate Seal

MTP
Lyophilized
Lyophilized
LiquidLiquidLiquidLiquidTabletLiquidLiquidLiquid

*1 …. .

MTP

*1Quantity

Main Ingredient1 plate (96-well)
1 vial (25 ng/vial)
1 vial1 vial (6 mL)
1 tube (0.2 mL)
1 bottle (12 mL)
1 bottle (26 mL)
2 tablets
1 bottle (12 mL)
1 bottle (10 mL)
1 bottle (50 mL)

Goat Anti-Rabbit IgG
Synthetic GLP-1 (7-36) amide
Biotinylated GLP-1 (7-36) amide
Rabbit anti-GLP-1 (7-36) amide
HRP-Labeled Streptoavidin
Phosphate buffer
0.015% Hydrogen peroxideo-Phenylenediamine dihydrochloride
1M H2SO4Phosphate buffer
Concentrated saline

3 sheets

Microtiter plate

MATERIALS REQUIRED BUT NOT PROVIDED•••••••

Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 490 nm
Rotator for microtiter plate
Washing device for microtiter plate and dispenser with an aspiration system
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Test tubes for preparation of Calibrator Solution
Graduated cylinder (1,000 mL)
Distilled water or de-ionized water

PRECAUTIONS
Protect reagents from strong light (e.g. direct sunlight) during storage and assay.
Satisfactory performance of the test is guaranteed only when reagents are used from a kit with identical lot number.
As pipetting operations may affect the precision of the assay, precisely pipette the prepared Calibrator Solutions orsamples into corresponding wells. Use a new tip for each sample and calibrator to avoid cross-contamination. Use clean
test tubes or vessels.
Always run a calibration curve when testing samples.

REAGENT PREPARATION
1. Preparation of Calibrator Solutions: Reconstitute the GLP-1 Calibrator (lyophilized GLP-1, 25 ng/vial) with 0.5 mL ofBuffer Solution, giving a 50 ng/mL Calibrator Solution after reconstitution. 0.1 mL of the reconstituted Calibrator
Solution is diluted with 0.2 mL of Buffer Solution to yield a 16.67 ng/mL Calibrator Solution. Repeat the serial dilution
to make Calibrator Solutions at 5.556, 1.852, 0.617, 0.206 ng/mL. Buffer Solution is used as the zero calibrator (0ng/mL).
Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels.
2. Preparation of Labeled Antigen: Reconstitute the Labeled Antigen with 6 mL of distilled water.
Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.
3. Preparation of SA-HRP Solution: Add 120 µL of SA-HRP into the bottle of SA-HRP Diluent and mix well.
Note: Diluted SA-HRP Solution must be prepared immediately before assay. Use clean test tubes or vessels.
4. Preparation of Substrate Solution: Dissolve one OPD Tablet in 12 mL of Substrate Buffer.
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Note: Substrate Solution must be prepared immediately before assay. Use clean test tubes or vessels.
5. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled or de-ionizedwater. Diluted Wash Solution is stable for 6 months at 4°C.
Note: During storage of the Wash Solution Concentrate at 4°C, precipitates may be observed, however, they will
dissolve when diluted.
6. Other reagents are ready for use.

STORAGEStore kit at 4°C. This kit can be used dividedly in strips of the plate. In such case, the rest of reconstituted reagents(calibrator and labeled antigen solutions) should be stored at 4°C and used within 2 weeks, or stored at or below -30°Cand used within 1 month.

SPECIMEN COLLECTION AND HANDLING
EDTA-2Na additive blood collection tube is recommended for plasma sample collection. Plasma samples must be usedas soon as possible after collection. If the samples are to be tested at a later time, they should be divided into test tubes insmall amounts and frozen at -30°C or below. Avoid repeated freeze/thaw cycles.

ASSAY PROTOCOL
1. Warm the reagents and samples to room temperature (20-30°C) at least 1 hour before beginning the test.
2. Add 350 µL/well of diluted Wash Solution into the wells and aspirate the Washing Solution. Repeat this washingprocedure twice, for a total of 3 washing steps. Finally, invert the plate and tap it onto an absorbent surface, such as
paper toweling, to ensure blotting free of most residual washing solution.
3. Add 40 µL of Labeled Antigen Solution into wells. Then add 30 µL of the prepared Calibrator Solutions (0, 0.206,0.617, 1.852, 5.556, 16.67, 50 ng/mL) or samples. Next, add 40 µL of GLP-1 Antibody into the wells.
4. Cover the plate with a Plate Seal and incubate at 4°C overnight (16-18 hours). Plate rotator is not needed for thisincubation.
5. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells 4 times with approximately 0.35 mL/wellof diluted Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, toensure blotting free of most residual washing solution.
6. Pipette 100 µL of diluted SA-HRP Solution into each of the wells.
7. Cover the plate with a Plate Seal and incubate at room temperature for 1 hour. During the incubation, the plateshould be rotated on a plate rotator.
8. Remove the Plate Seal, aspirate and wash the wells 5 times with approximately 0.35 mL/well of diluted Wash
Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting freeof most residual washing solution.
9. Add 100 µL of Substrate Solution (dissolved OPD tablet) into the wells, cover the plate with a Plate Seal and incubatefor 30 minutes at room temperature.
10. Add 100 µL of Stop Solution into the wells to stop the reaction.
11. Read the optical absorbance of the wells at 490 nm. The optical absorbance of reaction solution in wells should beread as soon as possible after stopping the color reaction.
Note:

Perform all determinations in duplicate.

RESULTS
Calculate mean absorbance values of wells containing the Calibrators and plot a calibration curve on semilogarithmic
graph paper (abscissa: concentration of Calibrators; ordinate: absorbance values of Calibrators). Use the calibration
curve to read GLP-1 concentrations in samples from the corresponding absorbance values.

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When a sample value exceeds 50 ng/mL, it must be diluted with Buffer Solution and re-assayed until the sample value iswithin the assay range.

PERFORMANCE
Typical Calibration Curve (example only, a new calibration curve for each run must be established by the end-user)

Analytical Recovery
GLP-1 AddedSample(ng/mL)
0Rat
0.5
Plasma
2.0
8.00
Human
0.5
Plasma2.0
8.0

Cross-Reactivity
Observed(ng/mL)
0.66
1.28
2.73
7.72
0.66
1.18
2.60
7.45

Precision and reproducibility• Intra-assay CV (%) Rat Plasma
Human Plasma•Inter-assay CV (%)

Rat Plasma
Human Plama

Expected(ng/mL)

Recovery(%)

1.16
2.66
8.66

110.4
102.6
89.20

1.16
2.66
8.66

101.7
97.7
86.0

Related Peptides
GLP-1 (1-37)
GLP-1 (1-36) amide
GLP-1 (7-37)
GLP-1 (7-36) amide
GLP-1 (9-36) amide

Cross-reactivity (%)
<0.1%
0.3%
<0.1%
100%
100%

5.36 – 6.60
4.69 – 10.67
5.51 – 18.87
9.63 – 17.57

Assay Range
0.206 – 50 ng/mL
Cross-Reactivity
No cross-reactivity with rat, human or mouse glucagons, human glicentin and rat, mouse, human GLP-2.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068 Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com
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